Introduction: Transforming growth factor (TGF)-β promotes the development of diabetic nephropathy by enhancing excessive production of extracellular matrix such as connective tissue growth factor (CTGF) in mesangial cells. However, the mechanism by which TGF-β induces CTGF expression has not been elucidated. In this study, we aimed to investigate contribution of small GTPase Rho and Rho-kinase to TGF-β-mediated CTGF expression in mesangial cells.

Methods and Results: Exposure of mouse mesangial cells (MES-13) to TGF-β induced activation of Rho and Rho-kinase. Y-27632, a specific Rho-kinase inhibitor, reduced TGF-β-mediated CTGF protein expression, indicating that TGF-β induces CTGF expression via Rho-kinase activation. Western blot analysis demonstrated that Y-27632 attenuates TGF-β-mediated phosphorylation of Erk1/2 and JNK/SAPK and nuclear translocation of NF-κB. Rho-kinase has been shown to regulate cytoskeleton reorganization. We therefore investigated whether Rho-kinase is involved in actin dynamics-induced NF-κB activation. Immunostaining studies demonstrated that Y-27632 and Cytochalasin D, an inhibitor of actin polymerization, attenuated TGF-β-mediated nuclear translocation of NF-κB, indicating that Rho-kinase-mediated actin reorganization regulates NF-κB activation. Furthermore, siRNA-mediated knockdown of Rho-kinase isoforms demonstrated ROCK2 but not ROCK1 is involved in these observations. In the present study, we demonstrate for the first time that Rho-kinase, especially ROCK2, mediates TGF-β-induced expression of CTGF via phosphorylation of Erk1/2 and JNK/SAPK and subsequent actin reorganization-mediated nuclear translocation of NF-κB in mesangial cells.

Conclusion: Rho-kinase could be an important therapeutic target against diabetic nephropathy.


Y. Nagai: None. D. Kawanami: None. K. Matoba: None. Y. Takeda: None. T. Akamine: None. S. Ishizawa: None. Y. Kanazawa: None. T. Yokota: None. K. Utsunomiya: None.

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